Fluorescent Complexes of Nucleic Acids/8-Hydroxyquinoline/Lanthanum(III) and the Fluorometry of Nucleic Acids

Abstract

The ternary fluorescent complexes of nucleic acids/8-hydroxyquinoline/ lanthanum (III) were studied. Nucleic acids in the study involve natured and thermally denatured calf thymus DNA, fish sperm DNA and yeast RNA. In the range of pH 8.0–8.4 (controlled by NH₃-NH₄Cl buffer) ternary fluorescent complexes are formed which emit at 485.0 nm for calf thymus DNA and at 480.0 nm for yeast RNA (when excited at 267.0 nm) and emits at 483.0 nm for fish sperm DNA when excited at 265.0 nm. Based on the fluorescence reactions sensitive fluorometric methods for nucleic acids were proposed. Using optimal conditions, the calibration curves were linear in the range of 0.4–3.6 μg˙ml^?1 for calf thymus DNA, 0.4–4.0 μg-ml^?1 for fish sperm DNA and 0.4–4.0 μg˙ml^?1 for yeast RNA, respectively. The limits of determination (3σ) were 0.076 μg˙ml^?1 for calf thymus DNA, 0.068 μg˙ml^?1 for fish sperm DNA and 0.329 μg˙ml^?1 for yeast RNA, respectively. Five synthetic samples were determined with satisfaction.

     

Determination of nucleic acids using calcein-neodymium complex as a fluorescence probe.

A novel fluorometric method has been developed for rapid determination of DNA and RNA with calcein-neodymium complex as a fluorescence probe. The method is based on the fluorescence enhancement of calcein-Nd(III) complex in the presence of DNA or RNA, with maximum excitation and emission wavelength at 489 nm and 514 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.5 - 3.0 microg/ml for both DNA and yeast RNA, 0.4 - 2.0 microg/ml for fish sperm DNA (FS DNA) and 0 - 3.0 microg/ml for calf thymus DNA (CT DNA). The corresponding detection limits are 15.1 ng/ml for DNA, 21.2 ng/ml for yeast RNA, 10.5 ng/ml for FS DNA and 8.9 ng/ml for CT DNA. The interaction mechanism for the binding of calcein-Nd(III) complex to DNA is also studied. The results of absorption spectra, fluorescence polarization measurements and thermal denaturation experiments, suggested that the interaction between calcein-Nd(III) complex and DNA is an electrostatic interaction.     

Fluorimetric determinations of nucleic acids using iron, osmium and samarium complexes of 4,7-diphenyl-1,10-phenanthroline

New sensitive, reliable and reproducible fluorimetric methods for determining microgram amounts of nucleic acids based on their reactions with Fe(II), Os(III) or Sm(III) complexes of 4,7-diphenyl-1,10-phenanthroline are proposed. Two complementary single stranded synthetic DNA sequences based on calf thymus as well as their hybridized double stranded were used. Nucleic acids were found to react instantaneously at room temperature in Tris-Cl buffer pH 7, with the investigated complexes resulting in decreasing their fluorescence emission. Two fluorescence peaks around 388 and 567 nm were obtained for the three complexes using excitation lambda(max) of 280 nm and were used for this investigation. Linear calibration graphs in the range 1-6 microg/ml were obtained. Detection limits of 0.35-0.98 microg/ml were obtained. Using the calibration graphs for the synthetic dsDNA, relative standard deviations of 2.0-5.0% were obtained for analyzing DNA in the extraction products from calf thymus and human blood. Corresponding Recovery% of 80-114 were obtained. Student's t-values at 95% confidence level showed insignificant difference between the real and measured values. Results obtained by these methods were compared with the ethidium bromide method using the F-test and satisfactory results were obtained. The association constants and number of binding sites of synthetic ssDNA and dsDNA with the three complexes were estimated using Rosenthanl graphic method. The interaction mechanism was discussed and an intercalation mechanism was suggested for the binding reaction between nucleic acids and the three complexes.     

Preparation and application of cysteine-capped ZnS nanoparticles as fluorescence probe in the determination of nucleic acids

Cysteine-capped ZnS nanometer-sized fluorescent particles were produced by a colloidal aqueous synthesis. The functionalized nanoparticles are water-soluble and suitable for biological application. A synchronous fluorescence method has been developed for the rapid determination of DNA with functionalized nano-ZnS as a fluorescence probe, based on the synchronous fluorescence enhancement of cysteine-capped nano-ZnS in the presence of DNA. When Deltalambda =190 nm, maximum synchronous fluorescence is produced at 267 nm at pH 5.12. Under optimum conditions, the synchronous fluorescence intensity is proportional to the concentration of nucleic acids in the range 0.1-1.2 microg ml(-1) for calf thymus DNA, 0.1-0.6 microg ml(-1) for fish sperm DNA. The corresponding detection limit is 32.9 ng ml(-1) for calf thymus DNA and 24.6 ng ml(-1) for fish sperm DNA. This method is simple, inexpensive, rapid and sensitive. The recovery and relative standard deviation are satisfactory.     

Determination of nucleic acids using fluorescence probe sensitized by microemulsion

Because the fluorescence of azur A can be quenched by adding nucleic acid, a sensitive fluorometric method for determination of nucleic acids at nanogram levels was established. Using optimal conditions, the calibration curves were linear in the range of 0-6.0 microg/mL for calf thymus deoxyribonucleic acid (ct DNA) and 0-7.0 microg/mL for herring sperm DNA (hs DNA). The limits of determination were 3.5 and 3.8 ng/mL, respectively, which shows the high sensitivity of this method. Triton X-100 microemulsion was applied as a sensitive media to enhance the sensitivity. The binding mode concerning the interactions of azur A with nucleic acids was also studied and the association constant with different binding numbers was obtained. The method has been applied to the determination of nucleic acid in both synthetic and real samples, such as cauliflower and pork liver, with satisfactory results.     

The determination of DNA based on light-scattering of a complex formed with histone

The interaction of histone with nucleic acids was characterized by light-scattering measurement using a common spectrofluorometer. Thereby, a sensitive and convenient method for the determination of nucleic acids was established. At pH 4.5-6.5, the interaction of histone with nucleic acids resulted in considerable light-scattering , and four characteristic peaks at 298, 450, 503, and 551 nm were observed. The light-scattering was applied to the determination of nucleic acids. The experiments indicated that, under optimal conditions, a linear relationship was obtained between the light-scattering intensity (I(LS)) and the concentration of nucleic acids. The linear ranges were 0.02-2.0 mug ml(-1) for fish sperm DNA (fsDNA), 0.05-1.5 mug ml(-1) for calf thymus DNA (ctDNA), 0.05-2.5 mug ml(-1) for Herring testis DNA (HtDNA), and 0.05-1.5 mug ml(-1) for human placenta DNA (hpDNA). The detection limits were 2.0 ng for fish sperm DNA, 2.0 ng for calf thymus DNA, 5.0 ng for Herring testis DNA, and 3.0 ng for human placenta DNA. The nucleic acids in yeast cell extraction were determined by simple vortex extraction. The results were satisfactory, and the recovery rates were in the range of 88-108%.     

Nephelometric determination of micro amounts of nucleic acids with protamine sulfate

Nucleic acids can form large particle complexes with protamine sulfate by electrostatic forces, which results in strong light scattering. Based on this, a nephelometric method is described for sensitive and convenient determination of nucleic acids with protamine sulfate by using a common spectrofluorimeter. Maximum light scattering is produced in the range of pH 2.2-4.4 with the same excitation and emission wavelengths at 365 nm. Under optimal conditions, the calibration curves are linear in the range 0.05-60.0 micrograms cm-3 for nucleic acids. The corresponding detection limits are 12.5 ng cm-3 for calf thymus DNA, 9.0 ng cm-3 for fish sperm DNA, and 18.0 ng cm-3 for yeast RNA, respectively. Six synthetic samples are determined with satisfactory results. The relative standard deviation of five replicate measurements is 3.2% for 2.0 micrograms cm-3 calf thymus DNA.     

Resonance Rayleigh scattering study of the reaction of nucleic acids with thionine and its analytical application

Resonance Rayleigh scattering (RRS) of the thionine (TH)-nucleic acids system and its analytical application have been studied. In pH 2.2 acidic buffer medium, some nucleic acids can react with TH to form TH-nucleic acids complex. This results in a great enhancement of RRS and the appearance of new RRS spectra. The RRS spectral characteristics of TH-ctDNA system, the affecting factors and the optimum conditions of the reaction have been investigated. The enhancement of the RRS signal is directly proportional to the concentration of nucleic acids in the range 0-10.0 microg/ml for calf thymus DNA and 0-15.0 microg/ml for yeast RNA, and its detection limits (3sigma) are 3.5 ng/ml for calf thymus DNA and 4.9 ng/ml for yeast RNA, respectively. The method shows a wide linear range and high sensitivity, and was applied to the determination of trace amounts of nucleic acid in synthetic samples and practical samples with satisfactory results. The bind properties for the interactions of TH with ctDNA were investigated using a Scatchard plot based on the measurement of the enhanced RRS data at 340 nm, and the binding number and intrinsic binding constant are 4.9 and 2.6 x 10(5) mol/dm(3), respectively.     

Determination of nucleic acids based on shifting the association equilibrium between tetracarboxy aluminum phthalocyanine and poly-lysine

A new method based on near-infrared (near-IR) fluorescence recovery was presented for the determination of nucleic acids. This method employed a two-reagent system composed of anionic tetracarboxy aluminum phthalocyanine (AlC4Pc) and polycationic poly-lysine. The fluorescence of AlC4Pc, with the maximum excitation and emission wavelengths at 620 and 701 nm, respectively, was quenched by poly-lysine with a proper concentration, but recovered by adding nucleic acids. Under optimal conditions, the recovered fluorescence was in proportional to the concentration of nucleic acids. The linear ranges of the calibration curves were 5-200 ng mL(-1) for both calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) with the detection limit of 2.6 ng mL(-1) for ctDNA and 2.1 ng mL(-1) for fsDNA. The relative standard deviation (n = 6) was 1.9 and 1.3% for 50 ng mL(-1) ctDNA and fsDNA, respectively. The proposed method was applied to the determination of nucleic acids in synthetic samples with satisfactory results.     

Fluorescence enhancement method for the determination of nucleic acids using cationic cyanine as a fluorescence probe

A fluorescence enhancement method with a cationic cyanine as a probe was developed for the determination of nucleic acids. Under the experimental conditions, the fluorescence enhancement of cyanine (lambda(ex)/lambda(em)= 524/591.5 nm) was observed in the presence of DNA. The calibration graphs were linear over the range of 0.01-15 microg mL(-1) for both calf thymus DNA (CT DNA) and fish sperm DNA (FS DNA). The limits of detection were 0.005 and 0.007 microg mL(-1) for CT DNA and FS DNA, respectively. The method was applied to the determination of DNA in synthetic and real samples and satisfactory results were obtained. A possible fluorescence enhancement mechanism was also studied.     

Determination of nucleic acids based on shifting the association equilibrium between tetrasulfonated aluminium phthalocyanine and acridine orange

Based on the ability of nucleic acids to shift the association equilibrium of the ion-association complex of Acridine Orange and tetrasulfonated aluminium phthalocyanine, thus leading to an increase in the phthalocyanine fluorescence, a method is suggested for the fluorimetric determination of nucleic acids. Investigations were carried out on the spectral characteristics, order of addition of reagents, selection of the buffer system, effect of pH, influence of reaction time, effect of salt, the usage of reagents, interference of foreign substances and the effect of different acridine derivatives. Under the optimum conditions, the calibration graphs for the determination of calf thymus DNA (CT DNA), salmon DNA (SM DNA) and yeast RNA were linear over the ranges 0.04-1.2, 0.04-1.2 and 0.1-1.2 micrograms cm-1, respectively. The detection limits for CT DNA, SM DNA and RNA were 17, 24 and 98 micrograms cm-3, respectively. The relative standard deviation (n = 6) was within 4.6% for the detection of samples. The method was applied to the determination of Staphylococcus aureus DNA and the result was in agreement with that achieved by a UV method.     

A kinetic fluorometric method for the determination of nucleic acids using a ternary equilibrium system of nucleic acids—iron (III) tetracarboxy phthalocyanine-poly-lysine coupled with the oxidation reaction between hydrogen peroxide and dl-tyrosine

Using the oxidation reaction between hydrogen peroxide and dl-tyrosine as fluorescence indication, the evident tuning effect of nucleic acids on catalytic activity of mimetic enzyme iron (III) tetracarboxy phthalocyanine (FeC₄Pc) in the presence of poly-lysine was observed and studied. The oxidation reaction between hydrogen peroxide and dl-tyrosine with FeC₄Pc as catalyst gave an intensively fluorescent compound, which has an excitation wavelength of 325 nm and an emission wavelength of 418 nm. The fluorescence was quenched by a proper concentration of poly-lysine due to its association with FeC₄Pc and consequently the descent of the catalytic activity of FeC₄Pc, but recovered by addition of nucleic acids. Under optimal conditions, the recovered fluorescence is proportional to the concentration of nucleic acids. Based on the fact, a kinetic fluorescent method was developed for the determination of nucleic acids. The calibration graphs are linear over the range 10-2000 ng/mL both for fish sperm DNA (FS DNA) and calf thymus DNA (CT DNA). The corresponding detection limits are 1.04 ng/mL for FS DNA and 1.18 ng/mL for CT DNA, respectively. Four synthetic and three real nucleic acid samples were determined with satisfactory results.     

Determination of nucleic acids in acidic medium by enhanced light scattering of large particles

The large particle light scattering technique was first developed as a sensitive and convenient analysis method for microdetermination of nucleic acids by using a common spectrofluorometer. In 0.1 mol l(-1) HCl, H(2)SO(4), or HNO(3) solution, the nucleic acids can aggregate to form large particles whose dimensions are comparable to the wavelength of UV-Vis light. The large particles can result in very strong light scattering which is well proportional to the concentration of nucleic acids in the range of 0.06-100.0 mug ml(-1) for calf thymus DNA, 0.05-60.0 mug ml(-1) for fish sperm DNA, and 0.6-90.0 mug ml(-1) for yeast RNA. The detection limits (3sigma) are 18.0 ng ml(-1) for calf thymus DNA, 16.0 ng ml(-1) for fish sperm DNA, and 57.6 ng ml(-1) for yeast RNA, respectively. Six synthetic samples were determined with satisfactory results.     

中性红荧光探针法测定生物大分子核酸

中性红 (NR)是一种吩嗪染料 ,至今已有许多关于 NR与 DNA相互作用的报道[1～ 5] .李克安[4 ] 和黄承志等 [5]利用共振光散射技术分别在酸性 (p H=2 .3 )和中性 (p H=7.6～ 7.8)条件下 ,建立了以 NR为探针测定痕量 DNA的方法 .我们 [2 ,3]曾利用荧光光谱方法研究了在 p H=7.4条件下 NR与 DNA之间的相互作用 ,发现利用吖啶橙和 NR之间的能量转移现象可以测定 DNA,但检出限偏高 ,且由于使用两种染料试剂 ,操作较繁琐 .为了克服吖啶橙、NR能量转移分析法的不足 ,本文建立了在 p H=4.5的条件下以单一染料 NR为荧光探针测定痕量核酸的…     

Determination of nucleic acids with crystal violet by a resonance light-scattering technique

For the first time, Crystal Violet (CV) was used to determine nucleic acid concentrations using the resonance light-scattering (RLS) technique. Based on the enhancement of the RLS of CV by nucleic acids, a new quantitative determination method for nucleic acids in aqueous solutions has been developed. At pH 5.03 and ionic strength 0.005 mol kg-1, the interaction of CV with nucleic acids results in three characteristic RLS peaks at 344.0, 483.0 and 666.0 nm. With 4.0 x 10(-5) mol l-1 of CV, linear relationships were found between the enhanced intensity of RLS at 666.0 nm and the concentration of nucleic acids in the range 0-2.5 micrograms ml-1 for herring sperm DNA, 0-4.0 micrograms ml-1 for calf thymus DNA and 0-4.5 micrograms ml-1 for yeast RNA. The limits of determination were 13.8 ng ml-1 for herring sperm DNA, 36.8 ng ml-1 for calf thymus DNA and 69.0 ng ml-1 for yeast RNA. The assay is convenient, rapid, inexpensive and simple.     

Determination for micro amounts of nucleic acids by a resonance light scattering technique with dequalinium chloride

Based on the strong enhancement effect of nucleic acids on resonance light scattering of dequalinium chloride, the determination method for micro amounts of nucleic acids has been developed. Under the experimental conditions (5.0x10(-5) mol l(-1) dequalinium, pH 7.0, at room temperature) the linear range of this assay is 0.04-10.0 mug ml(-1) for calf thymus DNA and fish sperm DNA, and 0.04-35.0 mug ml(-1) for yeast RNA. The detection limits (3sigma) are 6.2 ng ml(-1) for calf thymus DNA, 7.4 ng ml(-1) for fish sperm DNA, and 7.0 ng ml(-1) for yeast RNA, respectively. Almost no interference can be observed from ionic strength, proteins, nucleoside, and most of the metal ions. Six synthetic samples were determined satisfactorily.     

Near-infrared fluorimetric determination of nucleic acids by shifting the ion-association equilibrium between tetracarboxy aluminum phthalocyanine and tetra-N-hexadecylpyridiniumyl porphyrin

A new method was developed for determination of micro amounts of nucleic acids based on near-infrared (near-IR) fluorescence recovery, employing a two-reagent system which is composed of an anionic tetracarboxy aluminum phthalocyanine (AlC₄Pc) and a cationic tetra-N-hexadecylpyridiniumyl porphyrin (TC₁₆PyP). The fluorescence of the AlC₄Pc, with the maximum emission wavelength at 701 nm, could be quenched by TC₁₆PyP at its proper concentration, but recovered by adding nucleic acids. Under optimal conditions, the recovered fluorescence is proportional to the concentration of nucleic acids. The calibration graphs are linear over the range of 1-200 ng mL⁻¹ for fish sperm DNA (FS DNA) and 2-400 ng mL⁻¹ for calf thymus DNA (CT DNA). The corresponding detection limits are 0.59 ng mL⁻¹ for FS DNA and 0.82 ng mL⁻¹ for CT DNA, respectively. Four synthetic and three real nucleic acid samples were determined with satisfactory results.     

Study on the resonance light scattering spectrum of berberine-cetyltrimethylammonium bromide system and the determination of nucleic acids at nanogram levels

The interaction of berberine with nucleic acid in the presence of cetyltrimethylammonium bromide (CTMAB) in aqueous solution has been studied by spectrophotometry and resonance light scattering (RLS) spectroscopy. At pH 7.30, the RLS signals of berberine were greatly enhanced by nucleic acid in the region of 300-600 nm characterized by four peaks at 324.0, 386.5, 416.5 and 465.0 nm. The binding properties were examined by using a Scatchard plot based on the measurement of enhanced RLS data at 416.5 nm. Under optimum conditions, the increase of RLS intensity of this system at 416.5 nm is proportional to the concentration of nucleic acid. The linear range is 7.5 x 10(-9)-7.5 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-2.5 x 10(-5) g ml(-1) for herring sperm DNA, and 5.0 x 10(-9)-2.5 x 10(-5) g ml(-1) for yeast RNA. The detection limits (S/N = 3) are 2.1 ng ml(-1) for calf thymus DNA, 6.5 ng ml(-1) for herring sperm DNA and 3.5 ng ml(-1) for yeast RNA, respectively. Three synthetic samples were analyzed satisfactorily.     

Cationic cyanine as a near-infrared fluorescent probe for the determination of nucleic acids

A new method with a cationic near-IR cyanine as fluorescent probe was developed for the determination of nucleic acids. The near-IR cyanine shows maximum excitation and emission wavelengths at 765 and 790 nm, respectively, in aqueous solution. The method is based on the fluorescence decrease of near-IR cyanine in the presence of nucleic acids. Under optimal conditions, the ratio of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.10-1.2 microg/mL for CT (calf thymus) DNA or SM (salmon sperm) DNA, and 0.10-1.6 microg/mL for yeast RNA. The detection limits were 30 ng/mL for CT DNA, 25 ng/mL for SM DNA and 70 ng/mL for yeast RNA. The relative standard deviation (n = 6) was 2.1% for 500 ng/mL CT DNA, 2.4% for 500 ng/mL SM DNA and 2.7% for 500 ng/mL yeast RNA, respectively.     

Cetyltrimethylammonium bromide sensitized resonance light-scattering of nucleic acid--Pyronine B and its analytical application

This is the first report on the determination of nucleic acids with Pyronine B (PB) sensitized by cetyltrimethylammonium bromide (CTMAB) with resonance light-scattering (RLS) technique. Under the experimental conditions (1 x 10(-5) mol l(-1) PB, 1 x 10(-5) mol l(-1) CTMAB, pH 7.4, at room temperature, ionic strength 0.02 mol l(-1) NaCl), the interaction of PB with DNA sensitized by CTMAB results in enhanced RLS signals at 328 and 377 nm in the enhanced regions. It was found that the enhanced RLS intensity at 328 nm was proportional to the concentration of DNA in the suitable ranges. The linear range of this assay is 0.0-1.2 microg ml(-1) for calf thymus, 0.0-0.8 microg ml(-1) for fish sperm DNA (fsDNA), and 0.04-1.4 microg ml(-1) for yeast RNA, respectively. The detection limits (3 sigma) are 6.1 ng ml(-1) for calf thymus DNA (ctDNA), 11.2 ng ml(-1) for fish sperm DNA, and 8.6 ng ml(-1) for yeast RNA, respectively. Six synthetic samples were determined satisfactorily. This method is simple, rapid and the dye is inexpensive and stable.